Cellular processes intimately tied to cancer and to a variety of other human pathologies, are globally regulated or coordinated by GTP and ATP, molecules that represent the energy currency of the cell and which are also the most common allosteric modulators of protein function. Methods to measure the levels of these molecules in vivo, with high temporal and spatial resolution, would be invaluable in understanding how variations in GTP and ATP concentrations regulate and coordinate cellular metabolic processes, and in elucidating the role played by disruption of cellular metabolism in cancer and other diseases. Such methods-luminescence by insect luciferase and fluorescence from GFP- ATP binding protein fusions-exist for ATP. The luciferase assay is widely used and has led to important recent discoveries regarding the role of variations in ATP levels in the cell cycle and human disease, including cancer. However, no equivalent methods exist for GTP, even though GTP, through its action on numerous G-proteins, arguably plays a larger regulatory role in the cell than ATP. To address the need for such technology we will: (1) Engineer insect luciferase so that it will specifically use GTP, rather than ATP, to generate light, and (2) Engineer GFP-G-protein fusions that will exhibit altered excitation spectra upon binding GTP. These genetically encoded sensors will provide two complementary methods for monitoring GTP levels and GTP/GDP ratios inside living cells with high temporal and spatial resolution. PUBLIC HEALTH RELEVANCE: Cancer and many other human diseases, especially those associated with aging, often involve changes in cellular metabolism: alterations in the nature and concentrations of the molecules that supply the energy for cellular activity and dis-coordination in the network of the many cellular processes normally involved in maintaining cellular health. Insight into how metabolic processes are coordinated and regulated in a healthy cell can come from monitoring the levels of regulatory molecules, but this can be challenging to do in living cells. The proposed work will make it possible to monitor levels of GTP, one of the most important of these regulatory molecules, and will therefore create an invaluable tool for cancer and biomedical research.